Tn vaccine composition and method for alleviating inflammation

ABSTRACT

The present invention develops a vaccine composition against and treatment or prevention on inflammation and lung injury (particularly hyperoxia-induced lung injury) and progression of periodontitis. Tn immunization increases serum anti-Tn antibody titers, while it decreases lavaged protein and cytokines, and also decreases mean linear intercept and lung injury score. Furthermore, the improvement in lung injury is accompanied by a decrease in NF-κB activity.

FIELD OF THE INVENTION

The invention relates to the field of treatment of inflammation-relateddiseases. Particularly, the invention relates to use of TN immunogen inreducing cytokines and treating inflammation-related diseases.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 12, 2017, isnamed G4590-01300_SL.txt and is 1 KB in size.

BACKGROUND OF THE INVENTION

Tn antigen (GalNAc-a-O-Ser/Thr), a mucin-type O-linked glycan, is awell-established cell surface marker for tumors. Elevated levels arecorrelated with cancer progression and prognosis. Tn antigen is foundabnormally over-expressed in various cancers by inhibiting the furtherextension of glycosylation. The substrate specificity and specificmolecular chaperon of T-synthase core 1 β3-Gal-T-specific molecularchaperone (Cosmc) have also been demonstrated by previous studies. Thus,either a defective T-synthase or decreased expression of Cosmc couldprevent the extension of O-linked glycosylation of mucin, resulting inan apparently increased expression of Tn antigen. When further extensionof O-linked glycosylation is blocked, Tn antigen can also be furthermodified with sialic acid residue by alpha-2,6-sialyltransferase togenerate sialyl Tn (NeuAca6GalNAc-Ser/Thr, sTn). US 20030170249 andUS20070275019 provide a vaccine comprising: (a) a pharmaceuticallyeffective amount of a carbohydrate antigen found on said cancer cells,or a mimetic thereof; and (b) a pharmaceutically acceptable carrier. Thecarbohydrate antigen can be Tn or sialyl-Tn. US 20100278818 provides apharmaceutical composition comprising an antibody, directed against Tnantigen. U.S. Pat. No. 8,383,767 found that coupling a glycoantigen Tn,sTn, or GM3 with a protein carrier containing an immunoglobulin (Ig) Fcdomain and a cysteine-rich domain significantly improved itsantigenicity. Chiang et al. developed an anti-Tn vaccine, which inducesanti-Tn antibodies in mice with high specificity and high affinity usinglinear array epitope technology (H. L. Chiang, C. Y. Lin, F. D. Jan, Y.S. Lin, C. T. Hsu, J. Whang-Peng, L. F. Liu, S. Nieh, C. C. Lin, J.Hwang, A novel synthetic bipartite carrier protein for developingglycotope-based vaccines. Vaccine 30 (2012)7573-7581).

There are also reports that Tn is elevated in inflammatory tissues; theexpression of Tn is associated with the extent of inflammatory responseupon tissue damage. For example, Tn syndrome is characterized by thedetection of Tn antigen on blood cells of all lineages. Tn antigen canbe detected on the IgA1 hinge region in some IgA nephropathy patients.Additionally, Tn is known to express in chronic inflammatory tissuessuch as those from patients with rheumatoid arthritis andosteoarthritis. Elevated Tn expression has been observed ininflammation-inflicted tissue damage and found to be associated withmodulation of the host immune response.

Hyperoxia increases NF-κB translocation in fetal and adult lungfibroblasts and the production of proinflammatory mediators such astumor necrosis factor-α (TNF-α), interferon-γ, and interleukin-1β(IL-1β) (H. D. Li, Q. X. Zhang, Z. Mao, X. J. Xu, N. Y. Li, H. Zhang,Exogenous interleukin-10 attenuates hyperoxia-induced acute lung injuryin mice. Exp. Physiol. 100 (2015) 331-330; and C. J. Wright, P. A.Dennery, Manipulation of gene expression by oxygen: a primer frombedside to bench. Pediatr. Res. 66 (2009) 3-10). Prolonged exposure tohyperoxia leads to inflammation and acute lung injury. No effectivetherapies have yet been established.

SUMMARY OF THE INVENTION

The present invention provides a vaccine, comprising about 0.1 mg toabout 4 mg of a Tn immunogen per dose and a pharmaceutically acceptableadjuvant solution in a ratio of about 0.5 to about 2 (v/v) to about 0.5to about 2 (v/v). In one embodiment, the ratio of Tn immunogen to theadjuvant solution is about 1 (v/v):about 1 (v/v). In one embodiment, thevaccine comprises about 0.1 mg to about 2 mg of a Tn immunogen per dosein a therapeutically effective and pharmaceutically acceptable adjuvantformulation.

The present invention provides a method of inducing an immune responsein a subject to treat and/or prevent an inflammatory disease (e.g.inflammation), comprising administering a single dose vaccine comprisingabout 0.1 mg to about 2 mg of a Tn immunogen to the subject. In oneembodiment, the single dose vaccine comprises about 0.1 mg to about 2 mgof a Tn immunogen per dose and an adjuvant solution in a ratio of about0.5 to about 2 (v/v) to about 0.5 to about 2 (v/v).

The invention also provides a method of inducing an immune response in asubject to treat or prevent an inflammation disease, comprisingadministering about 0.1 mg to about 2 mg of a Tn immunogen per dose tothe subject at least four times at biweekly intervals. In oneembodiment, the method further comprises an additional immunization oneweek after the fourth immunization. In one embodiment, the additionalimmunization can be conducted one or more times after the lastimmunization. In one embodiment, the single dose vaccine comprises about0.1 mg to about 2 mg of a Tn immunogen per dose and an adjuvant solutionin a ratio of about 0.5 to about 2 (v/v) to about 0.5 to about 2 (v/v).

The inflammation disease is progression of periodontitis, organ injuryor organ fibrosis. In one embodiment, the organ injury is lung injury,renal injury or liver injury. In another embodiment, the lung injury ishyperoxia-induced lung injury.

The method of the present invention can reduce the levels ofinterleukine-6 (IL-6) and TNF-α and reduce the activity of NF-κB in acell or a subject. According to the invention, the cell or the subjecthas an elevated Tn expression and the Tn expression is upregulated byTNF-α and IL-6. Furthermore, the elevated Tn levels is commonlyregulated by the cytokine-Cosmc signaling axis.

The Tn immunogen can be conjugated with a carrier polypeptide at aweight ratio of about 3 to about 8:about 1. The carrier protein is anantigen presenting cell (APC) binding domain and or a cysteine-richdomain. In some embodiments, the cysteine-rich domain contains 6cysteine residues (SEQ ID NO: 1); preferably, the cysteine-rich domainhas the amino acid sequence of Pro-Cys-Cys-Gly-Cys-Cys-Gly-Cys-Gly-Cys(SEQ ID NO: 2). In another further embodiment, the cysteine-rich domaincontains 2 to 30 repeats of the amino acid sequence.

The Tn immunogen is N-acetyl galactosamine o-linked to serine orthreonine. The Tn immunogen is administered in the presence of about 0.2ml to about 2 ml adjuvant. The Tn immunogen is administered at a doseranging from about 0.1 mg to about 2 mg. The administration of the Tnimmunogen according to the method of the invention can produce anti-Tnantibody with high serum titers.

BRIEF DESCRIPTION OF THE DRAWING

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1(A) and (B) show serum titers of anti-Tn antibody before andafter immunization. The levels of anti-Tn antibody before immunization(pre) were low in all mice. In FIG. 1(A) the mice received carrierprotein developed low serum Tn antibody titers and in FIG. 1(B) the micereceived Tn immunization developed high serum antibody titers after thefirst immunization (621) and the antibody titers remained high after thesecond immunization (628).

FIG. 2 shows body weight at sacrifice. The mice exposed to the room airand hyperoxia all survived. The mice reared in O₂-enrich atmosphereexhibited significantly lower body weights than mice reared in room air(RA) at sacrifice (***P<0.001).

FIGS. 3A to C show bronchoalveolar lavage fluid (BALF) protein andcytokines levels. (A, B) The mice treated with carrier protein or Tnvaccine and exposed to hyperoxia exhibited significantly higher totalprotein and IL-6 levels in BALF than those exposed to room air (RA)(**P<0.01 and ***P<0.001). Tn immunization significantly lowered thehyperoxia-induced increase in the IL-6 level (***P<0.001). (C) The micetreated with carrier protein and exposed to hyperoxia exhibited asignificantly higher TNF-α level in BALF than those exposed to RA(**P<0.01). Tn immunization lowered the hyperoxia-induced increase inthe TNF-α level.

FIGS. 4(A) to (C) show (A) representative histology, (B) lung injuryscore, and (C) mean linear intercept (MLI) in mice treated with carrierprotein or Tn vaccine and exposed to RA or hyperoxia. The mice treatedwith carrier protein and exposed to hyperoxia exhibited a significantlyhigher lung injury score and MLI compared to the mice treated withcarrier protein or Tn vaccine and exposed to RA (***P<0.001). Tnimmunization significantly lowered the hyperoxia-induced increase in thelung injury score and MLI (***P<0.001).

FIGS. 5(A) to (C) show (FIG. 5A) representative Western blots and (FIG.5B) quantitative data determined using densitometry for nuclearfactor-κB (NF-κB) and (FIG. 5C) cytosol phospho-I-κBα in lung tissues.The mice treated with carrier protein and exposed to hyperoxia exhibitedsignificantly higher nuclear NF-κB p65 and cytosol phospho-IκBα levelscompared to the mice treated with carrier protein or Tn vaccine andexposed to RA (*P<0.05). Treatment with Tn vaccine significantly loweredthe hyperoxia-induced increase in the nuclear NF-κB p65 and cytosolphospho-IκBα (*P<0.05).

FIGS. 6A to C show that Tn levels is up-regulated in inflammatorytissues and cells. FIG. 6A. Immunohistochemical analysis of Tn antigenin inflammatory tissues (top) and normal tissues (bottom). Tn stainingin atherosclerotic aorta (left; labeled Aorta), bronchitis tissue(middle; labeled Bronchi), and periodontitis tissue (right; labeledGingiva). Brown color represents Tn antigen expression. FIG. 6B. HGFswere treated with conditioned media from U937 cells stimulated with LPS(0, 10, 30 and 100 ng/ml; 24 hours) for 24 hours and then stained withpurified rabbit anti-Tn antibody (in red) and DAPI (in blue). FIG. 6C.U937 cells were treated with LPS (0, 10, 30 and 100 ng/ml) for 24 hours,the secretions of TNF-α were analyzed by ELISA. Original magnifications(100×). Scale bar, 50 μm. FIG. 6D. U937 cells were treated with LPS (0,10, 30 and 100 ng/ml) for 24 hours, the secretions of IL-6 were analyzedby ELISA. Original magnifications (100×). Scale bar, 50 μm. FIG. 6E.U937 cells were treated with LPS (0, 10, 30 and 100 ng/ml) for 24 hours,the secretions of IL-1β were analyzed by ELISA. Original magnifications(100×). Scale bar, 50 μm.

FIGS. 7A and 7B show pro-inflammatory cytokines, TNF-α and IL-6,up-regulates Tn levels in HGFs. FIG. 7A. The effect of pro-inflammatorycytokines on Tn expression in HGFs. HGFs were treated with purifiedTNF-α, IL-6 and IL-1β at the concentration of 0, 10, 30, and 100 ng/mlfor 24 hours and then stained with purified rabbit anti-Tn antibody (inred) and DAPI (in blue). Original magnification (100×); scale bar, 50μm. FIG. 7B. Time course analysis of Tn expressions in HGFs after TNF-αtreatment. HGFs were treated with purified TNF-α at the concentration of30 ng/ml for 4, 8, 12, 24 and 48 hours and then stained with purifiedrabbit anti-Tn antibody (in red) and DAPI (in blue) (Magnifications630×; scale bar, 50 μm). The experiments were repeated at least threetimes.

FIGS. 8A to D show that TNF-α up-regulated Tn levels is throughdown-regulation of the COSMC gene in HGFs. FIG. 8A. Effect of TNF-α anddemethylation agents (5-aza-dC) on mRNA expression of COSMC andT-synthase in HGFs. qPCR was used to analyze COSMC and T-synthase mRNAexpression in HGFs upon TNF-α and 5-aza-dC treatment. The mRNAexpression of COSMC and T-synthase was normalized to GAPDH andstatistically analyzed. (*: p<0.05 compared with TNF-α only)Calculations of relative gene expression (normalized to GAPDH referencegene) were performed according to the ΔΔCT method. Fidelity of the PCRreaction was determined by melting temperature analysis. FIG. 8B.Western blotting was used to analyze the protein levels of Cosmc andT-synthase upon 6 and 24 hours of TNF-α treatment. FIG. 8C. and FIG. 8D.HGFs were treated with purified TNF-α for 6 or 24 hours and thenimmunofluorescent stained with anti-Cosmc (in red) and anti-T-synthaseantibodies (in green) and DAPI (in blue). Original magnification (100×);scale bar, 50 μm.

FIGS. 9A and B show that TNF-α-induced COSMC gene hypermethylation andTn expression can be suppressed by demethylation agents (5-aza-dC). FIG.9A. Effect of TNF-α and demethylation agents on the methylation level ofCOSMC gene in HGFs. The comparison of methylation changes is in the HGFswhich were treated with or without TNF-α and demethylation agents. TheUCSC genome browser illustrated the orientation and the first exon ofCOSMC (blue bar), GC percent (black scale bar), CpG islands (green bar)and the sequencing region of bisulfite pyrosequencing (COSMC_py02, blackbar). The red circle shows CG islands, and the black color displays thelevel of methylation. FIG. 9B. HGFs were co-treated with purified TNF-αand different concentrations of 5-aza-dC (0, 1, 2 and 5 μM) for 24 hoursand then immunofluorescent stained with rabbit anti-Tn antibody (in red)and DAPI (in blue). Magnification 630×; Scale bar, 50 μm.

DETAILED DESCRIPTION OF THE INVENTION

Several aspects of the invention are described below with reference toexample applications for illustration. It should be understood thatnumerous specific details, relationships, and methods are set forth toprovide a full understanding of the invention. One having ordinary skillin the relevant art, however, will readily recognize that the inventioncan be practiced without one or more of the specific details or withother methods. The present invention is not limited by the illustratedordering of acts or events, as some acts may occur in a different orderand/or concurrently with other acts or events.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting with respect to theinvention. As used herein, the singular forms “a”, “an” and “the” areintended to include the plural forms as well, unless the context clearlyindicates otherwise.

As used herein, the term “expression” is defined as the transcriptionand/or translation of a particular nucleotide sequence driven by itspromoter.

As used herein, the term “promoter” is defined as a DNA sequencerecognized by the synthetic machinery of the cell, or introducedsynthetic machinery, required to initiate the specific transcription ofa polynucleotide sequence.

As used herein, the term “antigen” or “Ag” is defined as a molecule thatprovokes an immune response. This immune response may involve eitherantibody production, or the activation of specificimmunologically-competent cells, or both. The skilled artisan willunderstand that any macromolecule, including virtually all proteins orpeptides, can serve as an antigen.

As used herein, “Tn antigen” denotes GalNAca-O-Ser/Thr, i.e. an antigenwherein the GalNAc residue is alpha-linked directly to the hydroxylgroup of a serine or threonine residue of a polypeptide chain expressedintracellularly or at the cell surface.

As used herein, the term “antibody” refers to an immunoglobulin moleculewhich specifically binds with an antigen. Antibodies can be intactimmunoglobulins derived from natural sources or from recombinant sourcesand can be immunoreactive portions of intact immunoglobulins. Antibodiesare typically tetramers of immunoglobulin molecules. The antibodies inthe present invention may exist in a variety of forms including, forexample, polyclonal antibodies, monoclonal antibodies, Fv, Fab andF(ab)₂, as well as single chain antibodies, human antibodies, andhumanized antibodies

As used herein, the term “polyclonal antibodies” refers to an antibodypopulation that includes a variety of different antibodies directed tothe same and/or to different epitopes within an antigen or antigens.

As used herein, the term “monoclonal antibody” refers to an antibodyobtained from a population of homogenous or substantially homogeneousantibodies. The term “monoclonal” is not limited to any particularmethod for making the antibody. Generally, a population of monoclonalantibodies can be generated by cells, a population of cells, or a cellline.

As used herein, the terms “patient,” “subject,” “individual,” and thelike are used interchangeably, and refer to any animal, or cells thereofwhether in vitro or in situ, amenable to the methods described herein.In certain non-limiting embodiments, the patient, subject or individualis a human

As used herein, the term “immunoglobulin” or “Ig” is defined as a classof proteins, which function as antibodies. Antibodies expressed by Bcells are sometimes referred to as the BCR (B cell receptor) or antigenreceptor. The five members included in this class of proteins are IgA,IgG, IgM, IgD, and IgE. IgA is the primary antibody that is present inbody secretions, such as saliva, tears, breast milk, gastrointestinalsecretions and mucus secretions of the respiratory and genitourinarytracts. IgG is the most common circulating antibody. IgM is the mainimmunoglobulin produced in the primary immune response in most subjects.It is the most efficient immunoglobulin in agglutination, complementfixation, and other antibody responses, and is important in defenseagainst bacteria and viruses. IgD is the immunoglobulin that has noknown antibody function, but may serve as an antigen receptor. IgE isthe immunoglobulin that mediates immediate hypersensitivity by causingrelease of mediators from mast cells and basophils upon exposure toallergen.

As used herein, the term “vaccine” refers to an immunogenic compositioncomprising an antigen which, when administered to a subject induces orstimulates or elicits cellular or humoral immune responses to theantigen of the vaccine. A vaccine may contain an adjuvant to produce amore robust immune response in the subject to the antigen.

As used herein, the term “adjuvant” refers to a substance used incombination with an antigen or combination of antigens to produce a morerobust immune response in a subject than the antigen or combination ofantigens alone.

As used herein, the phrases “stimulating an immune response”, “inducingan immune response” and “eliciting an immune response” are usedinterchangeably unless stated otherwise and include, but are not limitedto, inducing, stimulating, or eliciting a therapeutic or prophylacticeffect that is mediated by the immune system of a subject.

An “effective amount” as used herein, means an amount which provides atherapeutic or prophylactic benefit.

The vaccination with Tn antigen would inhibit NF-κB activity and inhibitinflammation through the action of anti-Tn antibody induced by Tnimmunization. The vaccine composition and method of the presentinvention can effectively treat or prevent an inflammation disease. Tnimmunization increases serum anti-Tn antibody titers, while decreasinglavaged protein and cytokines. The present invention thus proposes thatTn immunization can attenuate inflammation-related disorders and organinjury. The present invention develops a vaccine composition against andtreatment or prevention on inflammation and lung injury (particularlyhyperoxia-induced lung injury) and progression of periodontitis. Tnimmunization also decreases mean linear intercept and lung injury score.Furthermore, the improvement in lung injury is accompanied by a decreasein NF-κB activity.

In one aspect, the present invention provides a single dose vaccine,comprising about 0.1 mg to about 2 mg of a Tn immunogen per dose and anadjuvant solution in a ratio of about 0.5 to about 2 (v/v) to about 0.5to about 2 (v/v). In one embodiment, the ratio of the Tn immunogen tothe adjuvant solution is about 1 (v/v):about 1 (v/v).

In some embodiments, the dose of Tn immunogen ranges from about 0.01 mgto about 4 mg; including, for example, 0.01 mg, 0.02 mg, 0.03 mg, 0.04mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, or 0.09 mg to about 0.1 mg; or0.1 mg to about 1.5 mg, about 0.1 mg to about 1.2 mg, about 0.1 mg toabout 1 mg, about 0.1 mg to about 0.8 mg, about 0.1 mg to about 0.5 mg,about 0.2 mg to about 1.5 mg, about 0.2 mg to about 1.2 mg, about 0.2 mgto about 1 mg, about 0.2 mg to about 0.8 mg, about 0.2 mg to about 0.5mg, about 0.5 mg to about 2 mg, about 0.5 mg to about 1.5 mg, about 0.5mg to about 1.2 mg, about 0.5 mg to about 1 mg, about 0.5 mg to about0.8 mg or about 1.0 mg to about 2 mg; or about 1 mg to 3 mg; or about1.5 mg to about 2.5 mg; or about 1.6 to about 2.4 mg; or about 1.7 mg toabout 2.3 mg, or about 1.8 to about 2.2 mg, or about 1.9 to about 2.1mg, or about 2.0 mg; or a value that falls in the range between any ofthe two above-recited values. The volume of the adjuvant solution rangesfrom about 0.1 ml to 2 ml; including for example, 0.2 ml to about 1 ml,about 0.2 ml to about 0.8 ml or about 0.2 ml to about 0.6 ml or about0.4 ml to about 2 ml, or about 0.4 ml to about 1.6 ml, or about 0.4 mlto about 1.2 ml.

In another aspect, the present invention provides a method of inducingan immune response to treat and/or prevent an inflammation disease in asubject, comprising administering a single dose vaccine comprising about0.1 mg to about 4 mg of a Tn immunogen per dose to the subject. In oneembodiment, the single dose vaccine comprises about 0.1 mg to about 2 mgof a Tn immunogen per dose and an adjuvant solution in a ratio of about0.5 to about 2 (v/v) to about 0.5 to about 2 (v/v).

In one aspect, the present invention provides a method of inducing animmune response to treat or prevent an inflammation disease in asubject, comprising administering about 0.1 mg to about 4 mg of a Tnimmunogen per dose to the subject at least four times at biweeklyintervals. In one embodiment, the method further comprises an additionalimmunization with about 0.1 mg to about 4 mg of the Tn immunogen oneweek after the fourth immunization. In one embodiment, the additionalimmunization can be conducted one or more times after the lastimmunization. In one embodiment, the single dose vaccine comprises about0.1 mg to about 2 mg of a Tn immunogen per dose and an adjuvant solutionin a ratio of about 0.5 to about 2 (v/v) to about 0.5 to about 2 (v/v).In one embodiment, the single dose vaccine comprises about 0.1 mg toabout 3 mg of a Tn immunogen per dose and an adjuvant solution in aratio of about 0.5 to about 2 (v/v) to about 0.5 to about 2 (v/v). Inone embodiment, the single dose vaccine comprises about 0.1 mg to about4 mg of a Tn immunogen per dose and an adjuvant solution in a ratio ofabout 0.5 to about 2 (v/v) to about 0.5 to about 2 (v/v). In oneembodiment, the single dose vaccine comprises about 0.1 mg to about 2.5mg of a Tn immunogen per dose and an adjuvant solution in a ratio ofabout 0.5 to about 2 (v/v) to about 0.5 to about 2 (v/v). In oneembodiment, the single dose vaccine comprises about 0.1 mg to about 1.5mg of a Tn immunogen per dose and an adjuvant solution in a ratio ofabout 0.5 to about 2 (v/v) to about 0.5 to about 2 (v/v).

The administration of the Tn immunogen according to the method of theinvention can produce anti-Tn antibody with high serum titers than thetiter of the control. In one embodiment, the serum titer of the producedanti-Tn antibody is at least about 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5 or 6or more (or a value that falls in the range between any of the twoabove-recited numbers) folds higher than the titer of the control. The“control” used herein refers to carrier polypeptide.

In one embodiment, the Tn immunization reduces interleukine-6 (IL-6) andTNF-α levels or decreases NF-κB activity in a cell or a subject.

In one embodiment, the cell or the subject has an elevated Tnexpression. In a further embodiment, the Tn expression is upregulated byTNF-α and IL-6. In another further embodiment, the elevated Tn levels iscommonly regulated by the cytokine-Cosmc signaling axis.

In one embodiment, the inflammation disease is progression ofperiodontitis, organ injury or organ fibrosis. In a further embodiment,the organ injury is lung injury, renal injury or liver injury. In afurther embodiment, the lung injury is hyperoxia-induced lung injury. Ina further embodiment, the organ fibrosis is lung fibrosis, liverfibrosis or renal fibrosis.

In one embodiment, the method further comprises a step of having anadditional immunization one week after the fourth immunization.

In some embodiments, the Tn immunogen can be conjugated with a carrierpolypeptide. The Tn immunogen and the carrier polypeptide are at aweight ratio of about 3 to about 8:about 1; preferably, the weight ratiois about 5:about 1. In some embodiments, the polypeptide includes, butis not limited to, an antigen presenting cell (APC) binding domain and acysteine-rich domain. In some embodiments, the APC binding domain is animmunoglobulin (Ig) Fc fragment or a receptor-binding domain of a toxin.In a further embodiment, the APC binding domain is a receptor-bindingdomain of Pseudomonas exotoxin A, tetanus toxin, or cholera toxin. In afurther embodiment, the APC binding domain is a Fc fragment of a humanIg. In some other embodiments, the cysteine-rich domain contains afragment of 10 amino acid residues, at least 3 of which are cysteineresidues. In another further embodiment, the cysteine-rich domaincontains 6 cysteine residues. Preferably, the cysteine-rich domain hasthe amino acid sequence of Pro-Cys-Cys-Gly-Cys-Cys-Gly-Cys-Gly-Cys (SEQID NO: 2). In another further embodiment, the cysteine-rich domaincontains 2 to 30 repeats of the amino acid sequence. Preferably, thecysteine-rich domain contains 7 repeats of the amino acid sequence. Inanother embodiment, the Tn is linked to the cysteine residues via alinker (such as a linker containing a maleimide functional group; forexample, N-maleimide or N-succinimidyl-6-maleimidocaproate). In anotherfurther embodiment, the Tn immunogen conjugated with a carrierpolypeptide is Fc fragment-7 repeats ofPro-Cys-Cys-Gly-Cys-Cys-Gly-Cys-Gly-Cys (SEQ ID NO: 2)-Tn. Preferably,the Tn immunogen conjugated with a carrier polypeptide is Fc fragment-7repeats of Pro-Cys-Cys-Gly-Cys-Cys-Gly-Cys-Gly-Cys (SEQ ID NO:2)-N-maleimide-Tn or Fc fragment-7 repeats ofPro-Cys-Cys-Gly-Cys-Cys-Gly-Cys-Gly-Cys (SEQ ID NO:2)-N-succinimidyl-6-maleimidocaproate-Tn.

In one embodiment, the Tn immunogen is N-acetyl galactosamine o-linkedto serine or threonine, which has the following structure.

In one embodiment, the Tn immunogen is administered in the presence of0.2 ml to 2 ml adjuvant. In some embodiments, the adjuvant is aluminumhydroxide, aluminum phosphate, calcium phosphate hydroxide, killedbacteria Bordetella pertussis, Mycobacterium bovis, toxoids, squalene,Quil A, saponins, IL-1, IL-2, IL-12, Freund's complete adjuvant orFreund's incomplete adjuvant. In a further embodiment, the adjuvant isaluminum phosphate.

Vaccine compositions comprising the Tn immunogen of the invention may beadministered to a subject already suffering from inflammation. Intherapeutic applications, compositions are administered to a patient inan amount sufficient to elicit an effective immune response to thepresent antigen and to cure or at least partially arrest symptoms and/orcomplications. An amount adequate to accomplish this is defined as“therapeutically effective dose.” Amounts effective for this use willdepend on, e.g., the peptide composition, the manner of administration,the stage and severity of the disease being treated, the weight andgeneral state of health of the patient, and the judgment of theprescribing physician, but generally the range for the initialimmunization (that is for therapeutic or prophylactic administration)can be from about 0.1 mg to 4 mg of Tn immunogen for a subject, followedby boosting dosage from about 0.1 mg to 4 mg of Tn immunogen pursuant toa boosting regimen described herein. In one embodiment, the initialimmunization is from about 0.1 mg to about 2 mg of Tn immunogen for asubject, followed by boosting dosage from about 0.1 mg to about 2 mg ofTn immunogen

The vaccine compositions are intended for parenteral, topical, nasal,oral or local administration. Preferably, the pharmaceuticalcompositions are administered parenterally, e.g., intravenously,subcutaneously, intradermally, or intramuscularly. Preferably, thevaccine is administered intramuscularly. The invention providescompositions for parenteral administration which comprise a solution ofthe vaccine compositions dissolved or suspended in an acceptablecarrier, preferably an aqueous carrier. These compositions may besterilized by conventional, well known sterilization techniques, or maybe sterile filtered. The resulting aqueous solutions may be packaged foruse as is, or lyophilized, the lyophilized preparation being combinedwith a sterile solution prior to administration. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents and the like, forexample, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, triethanolamineoleate, etc.

By way of example, and not of limitation, examples of the presentinvention shall now be given.

EXAMPLES

Materials and Methods

Animal Model

Five-week-old female C57BL/6NCrlBltw mice were obtained from BioLASCOTaiwan Co., Ltd and were maintained in a pathogen free facility. Animalswere kept at approximately 25° C. and pelleted food and water wereavailable ad libitum throughout the experiment. The study protocol wasapproved by the Institutional Animal Care and Use Committee of TaipeiMedical University (LAC-2016-0047). As described herein, an animal modelwas used as a demonstration of vaccine efficacy. Human equivalent dosageinformation can be readily calculated by those of skill in the art andis described in, for example, Herati et al, Cold Springs HarborPerspectives in Biology, Mar. 17, 2017.

Preparation of Tn Vaccine

Tn vaccine was prepared by conjugating Tn to a carrier protein asdescribed in previous study (H. L. Chiang, C. Y. Lin, F. D. Jan, Y. S.Lin, C. T. Hsu, J. Whang-Peng, L. F. Liu, S. Nieh, C. C. Lin, J. Hwang,A novel synthetic bipartite carrier protein for developingglycotope-based vaccines. Vaccine 30 (2012) 7573-7581). Tn wasconjugated to ratFc(Cys42)Histag2 or GST(Cys6)Histag2 at aglycotope/carrier protein weight ratio of 5 to 1. Conjugation wasperformed in buffer containing 20 mM sodium phosphate, pH 7.9, 8 M urea,500 mM imidazole, and 0.2 mM TCEP. After 48 hours, conjugate wasrefolded against phosphate-buffered saline (PBS) with 0.2 mM TCEP.GST(Cys6) was dialyzed against PBS with 0.2 mM TCEP. Differentglycotopes and Linker (N-Succinimidyl-6-Maleimidocaproate) wereconjugated to GST(Cys6) at 4° C. for 48 hours.

Experimental Groups of Mice

Five-week-old female C57BL/6NCrlBltw mice were subcutaneously immunizedwith Tn vaccine at a dose of 20 μg or carrier protein (10 μg ofmFc(Cys42-Tn)Histag2) in the presence of adjuvant in 100 μl for fourtimes at biweekly intervals and one additional immunization at one weeklater after the fourth immunization. Blood was withdrawn from facialvein for anti-Tn antibody titer measurement using enzyme-linkedimmunosorbent assay (ELISA) on days 0, 42, and 49. Four days after thelast immunization, mice were exposed to room air (RA) or oxygen-enrichedatmosphere (100% O₂) for up to 96 hours. Oxygen exposures was carriedout in a transparent 60×50×40-cm Plexiglas chamber into which oxygen wascontinuously delivered at 4 l/min and oxygen levels were monitored witha ProOx Model 110 monitor (NexBiOxy, Hsinchu, Taiwan) and humidity waschecked daily and the value was 60-80%. We obtained four study groups asfollows: carrier protein+RA (n=6), Tn vaccine+RA (n=6), carrierprotein+O₂ (n=6), and Tn vaccine+O₂ (n=5). Mice were deeply anesthetizedwith an overdose of isoflurane after 96 hours O₂ treatment. Lung waslavaged with 0.6 ml 0.9% saline at 4° C. which washed in and out of thelungs three times and then recovered. This washing procedure wasrepeated two more times for each animal, with the three washes beingpooled, and the total volume recorded. The right lung was ligated, andleft lung was fixed by tracheal instillation of 4% bufferedparaformaldehyde at a pressure of 25 cm H₂O for 10 min afterbronchoalveolar lavage.

Analysis the Levels of Serum Anti-Tn Antibody by ELISA

GST(Cys6-Tn) was coated on 96-well flat-bottomed plates (Falcon Labware,Lincoln Park, N.J., USA) at a concentration of 1.5 μg/ml. Variousdiluted antiserum was add to each coated well. After incubating at 37°C. for 2 hours, the wells were washed with PBS three times.Subsequently, peroxidase-conjugated anti-human immunoblobulin was added,and the plates were incubated at 37° C. for 1 hour. The substratesolution contained 0.54 mg/ml2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) and 0.01% H₂O₂,and 0.1M citric acid (pH 4.2). Absorbance was read at 410 nm.

Bronchoalveolar Lavage Fluid Protein and Cytokines Analysis

Total protein concentration in bronchoalveolar lavage fluid (BALF) wasmeasured with a bicinchoninic acid assay (Pierce Chemical, Rockford,Ill., USA). The levels of IL-6 and TNF-α in the BALF were determinedusing the ELISA kit (Cloud-Clone Corp., Houston, Tex., USA). The datawere expressed in mg/ml and pg/ml, respectively.

Western Blot Analysis of NF-κB

Subcellular protein fractionation was done using the Subcellular ProteinFractionation Kit for Tissue (Thermo Scientific, Melbourne, VIC,Australia, ca t#87790). Nuclear protein extracts were used to detect theNF-κB p65 (SC-372, Santa Cruz Biotechnologies, Santa Cruz, Calif., USA)subunit and PCNA (SC-7907); cytoplasmic protein extracts were used todetect IκB-α (SC-1643) and β-actin (SC-47778). Protein concentrationswere determined with a bicinchoninic acid protein assay kit. Proteinswere separated on a 12% sodium dodecyl sulfate polyacrylamide gel andtransferred onto polyvinylidene difluoride membranes, and the membraneswere blocked in 5% skim milk at room temperature for 1 hour. Themembranes were incubated at 4° C. overnight with antibodies.Subsequently, the membranes were incubated with HRP-conjugated secondaryantibody at room temperature for 1 hour. The signal was visualized byenhanced chemiluminescence reagents according to the manufacturer'sprotocol. Antibodies to β-actin and PCNA were used as internal controlsof nuclear and cytosolic protein loading, respectively. All blottingexperiments were performed at least three times with different mice.

Lung Morphometry

To standardize analysis, sections are taken from the right middle lobeof the right lung. Five-μm lung tissue sections are stained withhematoxylin and eosin and assessed for lung morphometry. Mean linearintercept (MLI), an indicator of mean alveolar diameter, is assessed in10 nonoverlapping fields [18].

Histology

Lung tissues were fixed in 4% paraformaldehyde in phosphate buffer,embedded in paraffin, stained with hematoxylin and eosin, and examinedby a pathologist who was blinded to the protocol and experimentalgroups. Lung injury was scored according to the following fourcriteria: 1) alveolar congestion, 2) hemorrhage, 3) infiltration ofneutrophils in the air space or vessel wall, and 4) thickness of thealveolar wall. Each item was graded according to a five-point scale asfollows: 0 for minimal (little) damage, 1 for mild damage, 2 formoderate damage, 3 for severe damage, and 4 for maximal damage [19].

Immunohistochemistry of NF-κB

After a routine deparaffinization step, heat-induced epitope retrievalwas performed by immersing the slides in 0.01 mol/L sodium citratebuffer (pH 6.0). To block the endogenous peroxidase activity andnonspecific antibody binding, sections were first preincubated for 1hour at room temperature in 0.1 mol/L PBS containing 10% normal goatserum and 0.3% H₂O₂ before incubating for 20 hours at 4° C. with therabbit polyclonal anti-NF-κB P65 (1:50 dilution; Abcam Inc., Cambridge,Mass., USA) as primary antibody. The sections were then treated for 1hour at room temperature with biotinylated goat anti-rabbit IgG (1:200,Vector, Calif., USA). This was followed by reaction with the reagentsfrom an ABC kit (Avidin-Biotin Complex, Vector, Calif., USA) accordingto the manufacturer's recommendations, and the reaction products werevisualized by diaminobenzidine substrate kit (Vector, Calif., USA). Allimmunostained sections were viewed and photographed by Olympus BX 43.

Statistical Analysis

All data are presented as mean±SD. Statistical analyses were performedusing one-way analysis of variance with a Tukey post hoc test formultiple group comparisons. Differences were considered statisticallysignificant when P<0.05.

EXAMPLES Example 1 Serum Titers of Anti-Tn Antibody

The levels of anti-Tn antibody were low in all the mice beforeimmunization and we count it as background (FIG. 1). The mice thatreceived carrier polypeptide (i.e., Fc fragment-7 repeats ofPro-Cys-Cys-Gly-Cys-Cys-Gly-Cys-Gly-Cys-N-succinimidyl-6-maleimidocaproate)and were housed in room air or exposed to hyperoxia showed backgroundserum anti-Tn antibody levels (FIG. 1A), while mice received Tnvaccination developed high serum anti-Tn antibody titers after Tnvaccination and anti-Tn antibody levels remained high after severalmonths of vaccination (FIG. 1B).

Example 2 Survival and Body Weight

The mice exposed to room air or hyperoxia all survived throughout thestudy period. The mice exposed to hyperoxia exhibited significantlylower body weights at sacrifice than those reared in room air (FIG. 2).

Example 3 Bronchoalveolar Lavage Fluid Protein and Cytokines Analysis

The mice treated with carrier protein, followed by exposure to hyperoxiaexhibited significantly higher total protein and IL-6 levels in BALFthan those exposed to room air (FIGS. 3A and B). On the other hand, themice treated with Tn vaccine and exposed to hyperoxia exhibited asignificantly lower IL-6 level in BALF than those treated with carrierprotein (FIG. 3B). The mice treated with carrier protein and exposed tohyperoxia exhibited a significantly higher TNF-α level in BALF thanthose exposed to RA (FIG. 3C). The mice treated with Tn vaccine andexposed to hyperoxia exhibited a lower TNF-α level in BALF. However, thedifference did not reach significance.

Example 4 Histology Results

Representative lung sections stained with hematoxylin and eosin frommice exposed to RA and hyperoxia are presented in FIG. 4A. Hyperoxiaresulted in inflammatory cells infiltration and simplification of thelung parenchyma, as indicated by greater linear intercept. The micetreated with carrier protein and exposed to hyperoxia exhibited asignificantly higher lung injury score and MLI compared to the micetreated with carrier protein or Tn vaccine and exposed to RA (FIGS. 4Band 4C). Treatment with Tn vaccine significantly decreased thehyperoxia-induced increase in the lung injury score and MLI.

Example 5 Immunohistochemistry of NF-κB

The immunohistochemical staining of NFκB was found primarily in thecytoplasm of alveolar macrophages, but the immunoreactivity was alsodisplayed in the nuclei of alveolar macrophage and small number ofalveolar epithelial cells (FIG. 5A). The lung of the hyperoxia groupimmunized with carrier protein exhibited more intense NFκBimmunoreactivity than the control and Tn-treated hyperoxia groups.

Example 6 Western Blot Analysis for NF-kB and IkBα

The mice treated with carrier protein and exposed to hyperoxia exhibiteda significantly higher nuclear NF-κB p65 and cytosol phospho-IκBα levelscompared to the mice treated with carrier protein or Tn vaccine andexposed to RA (FIGS. 5B and 5C). The group of rats treated with Tnvaccine exhibited significantly levels of NFκB p65 and cytosolphospho-IκBα even in the hyperoxia-treated rats.

Example 7 Elevated Tn Levels in Inflammatory Tissues and Cells

To examine whether elevated Tn levels are associated with inflammation,Tn levels were measured in inflammatory tissues usingimmunohistochemistry (IHC). A significant increase in Tn levels wasobserved in tissues of atherosclerosis, bronchitis and periodontitis butnot in their corresponding normal tissues (FIG. 6A). To investigate thepossible regulation of Tn levels by inflammatory cytokines, conditionedmedia from monocyte U937 cells stimulated with LPS were used. Tn levelsin human gingival fibroblasts (HGFs) replenished with one-dayconditioned media from LPS-stimulated U937 cells were observed toincrease in an LPS-dose-dependent manner (FIG. 6B). The secretion ofinflammatory cytokines (ex: TNF-α, IL-6, and IL-1β) was significantlyhigher in conditioned media from U937 cells treated with LPS (10, 30, or100 ng/ml) for 24 hours compared with media from U937 cells culturedwithout LPS (FIG. 6C).

Example 8 TNF-α and IL-6 Up-Regulate Tn Expression in HGFs

To determine whether cytokine(s) can elevate Tn levels, HGFs weretreated with various amounts of purified cytokines. As shown in FIG. 7A,Tn levels in HGFs were most responsive to TNF-α, moderately responsiveto IL-6, and not responsive to IL-1β, even at a concentration of 100ng/ml under the experimental conditions. Elevation of Tn by TNF-α (30ng/ml) was shown to be time dependent. Tn levels in HGFs wereessentially unchanged upon 4 hrs of TNF-α treatment. Gradual increase inTn levels was observed between 8 to 12 hrs. The level of Tn graduallydecreased after 24 hours of TNF-α treatment and markedly decreased after48 hours of TNF-α treatment (FIG. 7B).

Example 9 TNF-α Up-Regulates Tn Expression Through Down-Regulation ofthe COSMC Gene

To explore the possible molecular mechanism underlying cytokine-mediatedup-regulation of Tn levels, the effect of TNF-α on the mRNA level of theCOSMC gene was investigated. As shown in FIG. 8A, TNF-α (100 ng/ml,treatment for 24 hours) significantly down-regulated the COSMC mRNA inHGFs. By contrast, TNF-α did not significantly alter the T-synthase mRNAlevel. Similar results were observed for the protein levels of Cosmc andT-synthase in HGFs upon TNF-α treatment (FIGS. 8B, 8C and 8D). Theeffect of TNF-α on the down-regulation of the COSMC gene could possiblyinvolve hypermethylation of the CpG islands in its promoter. Usingbisulfite pyrosequencing to quantify the methylation change in thepromoter of the COSMC gene, four CpG sites were significantlyhypermethylated by TNF-α treatment (FIG. 9A). Pretreatment of HGFs withdemethylating agents decreased the methylation of the four CpG sites inthe COSMC promoter in a dose-dependent manner (FIG. 9A), andcorrespondingly, increased the expression of the COSMC mRNA anddecreased the level of Tn (FIG. 9B). In the aggregate, our resultssuggest that cytokine-mediated up-regulation of Tn levels is due todown-regulation of COSMC, which involves hypermethylation of the COSMCgene promoter.

We claim:
 1. A method of inducing an immune response in a subject totreat or prevent hyperoxia-induced lung injury, comprising administeringto the subject a single dose of a vaccine, wherein the vaccine comprisesabout 0.1 mg to about 4 mg of a GalNAc-a-O-Ser/Thr (Tn) immunogen perdose and an adjuvant solution in a ratio of about 0.5 to about 2 (v/v),wherein the Tn immunogen is conjugated with a carrier polypeptideselected from: the amino acid sequence ofPro-Cys-Cys-Gly-Cys-Cys-Gly-Cys-Gly-Cys (SEQ ID NO:2), or Fc fragment-7repeats of (SEQ ID NO:2)-N-maleimide or Fc fragment-7 repeats of (SEQ IDNO:2)-N-succinimidyl-6-maleimidocaproate.
 2. The method of claim 1,wherein the about 0.1 mg to about 2 mg of a Tn immunogen per dose isadministered four times at biweekly intervals.
 3. The method of claim 2,wherein the method further comprises a step of having an additionalimmunization with about 0.1 mg to about 2 mg of the Tn immunogen oneweek after the fourth immunization.
 4. The method of claim 1, whereinthe administration of the single dose vaccine produces anti-Tn antibodywith high serum titers than the titer of the control.
 5. The method ofclaim 4, wherein the he serum titer of the produced anti-Tn antibody isat least 2 folds higher than the titer of the control.
 6. The method ofclaim 1, wherein interleukine-6 (IL-6) and TNF-α levels or the activityof NF-κB in a subject is reduced after treatment with the vaccine. 7.The method of claim 1, wherein the subject has an elevated Tnexpression.
 8. The method of claim 7, wherein the Tn expression isupregulated by TNF-α and IL-6.
 9. The method of claim 7, wherein theelevated Tn expression is commonly regulated by the cytokine-Cosmcsignaling axis.
 10. The method of claim 1, wherein the Tn immunogen andthe carrier polypeptide are at a weight ratio of about 3 to about8:about
 1. 11. The method of claim 1, wherein the Tn immunogen has thefollowing structure:


12. The method of claim 1, wherein the vaccine is-formulated as a singledose vaccine in a pharmaceutically acceptable formulation.